Regulation of blood glucose levels depends on the property of beta cells to couple glucose sensing with insulin secretion. This is accomplished by the concentration-dependent flux of glucose through glycolysis and oxidative phosphorylation, generating ATP. The resulting rise in cytosolic ATP/ADP inhibits K(ATP) channels, inducing membrane depolarization and Ca(2+) influx, which prompts insulin secretion. Evidence suggests that this coupling of glucose sensing with insulin secretion may be compartmentalized in the submembrane regions of the beta cell. We investigated the subcellular responses of key components involved in this coupling and found mitochondria in the submembrane zone, some tethered to the cytoskeleton near capillaries. Using Fluorescent Lifetime Imaging Microscopy (FLIM), we observed that submembrane mitochondria were the fastest to respond to glucose. In the most glucose-responsive beta cells, glucose triggers rapid, localized submembrane increases in ATP and Ca(2+) as synchronized ~4-min oscillations, consistent with pulsatile insulin release after meals. These findings are consistent with the hypothesis that glucose sensing is coupled with insulin secretion in the submembrane zone of beta cells. This zonal adaptation would enhance both the speed and energy efficiency of beta cell responses to glucose, as only a subset of the most accessible mitochondria would be required to trigger insulin secretion.